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5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside Renders Glucose Output by the Liver of the Dog Insensitive to a Pharmacological Increment in Insulin

Raul C. Camacho, D. Brooks Lacy, Freyja D. James, E. Patrick Donahue, and David H. Wasserman

Department of Molecular Physiology and Biophysics and Diabetes Research and Training Center, Vanderbilt University School of Medicine,

Nashville, TN 37232-0615

Running Title: AICAR stimulates hepatic glucose output Address Correspondence to:

Raul Camacho Ph.D. Department of Medicine, Division of Endocrinology, Belfer 701 Albert Einstein College of Medicine 1300 Morris Park Ave. Bronx, NY 10461 phone: (718) 430-3312 fax: (718) 430-8557 e-mail: rcamacho@aecom.yu.edu

Articles in PresS. Am J Physiol Endocrinol Metab (July 26, 2005). doi:10.1152/ajpendo.00247.2005

Copyright © 2005 by the American Physiological Society.

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ABSTRACT

This study aimed to test whether stimulation of net hepatic glucose output (NHGO)

by increased concentrations of the adenosine 5’-monophosphate analogue, 5-

aminoimidazole-4-carboxamide-1-β-D-ribosyl-5-monophospate, can be suppressed by

pharmacological insulin levels. Dogs had sampling (artery, portal vein, hepatic vein) and

infusion (vena cava, portal vein) catheters, and flow probes (hepatic artery, portal vein)

implanted >16 days prior to a study. Protocols consisted of equilibration (-130 to -30 min),

basal (-30 to 0 min) and hyperinsulinemic-euglycemic (0 to 150 min) periods. At t=0 min,

somatostatin was infused and basal glucagon was replaced via the portal vein. Insulin was

infused in the portal vein at either 2 (INS2) or 5 (INS5) mU•kg-1•min-1. At t=60 min, a 1

mg•kg-1•min-1 portal venous 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside

(AICAR) infusion was initiated. Arterial insulin rose ~9-fold and ~27-fold in INS2 and

INS5. Glucagon, catecholamines and cortisol did not change throughout the study. NHGO

was completely suppressed prior to t=60 min. Intraportal AICAR stimulated NHGO by 1.9

± 0.5 and 2.0 ± 0.5 mg•kg-1•min-1 in INS2 and INS5, respectively. AICAR stimulated

tracer-determined endogenous glucose production similarly in both groups. Intraportal

AICAR infusion significantly increased hepatic acetyl-CoA carboxylase (ACC, Ser79)

phosphorylation in INS2. Hepatic ACC (Ser79) phosphorylation, however, was not

increased in INS5. Thus, intraportal AICAR infusion renders hepatic glucose output

insensitive to pharmacological insulin. The effectiveness of AICAR in countering the

suppressive effect of pharmacological insulin on NHGO occurs even though AICAR-

stimulated ACC phosphorylation is completely blocked.

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Keywords: Hepatic glucose production, glycogenolysis, AICAR, AMPK, AMP, ZMP

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INTRODUCTION

Iatrogenic hypoglycemia due to hyperinsulinemia is the leading short-term

complication for both type 1 and type 2 diabetic patients striving to maintain tight

glycemic control (10). Current treatments for hypoglycemia include carbohydrate

ingestion, or if necessary (i.e. during unconsciousness), administration of exogenous

glucagon. However, depending on the degree of hyperinsulinemia, neither of these

treatments may be sufficient. A more optimal treatment would be one that is effective in

the presence of hyperinsulinemia.

The compound, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside

(AICAR), is taken up by cells and phosphorylated to become the adenosine 5’-

monophosphate (AMP) analogue, 5-aminoimidazole-4-carboxamide-1-β-D-ribosyl-5-

monophospate (ZMP). Intraportal AICAR infusion (1 mg•kg-1•min-1) stimulates hepatic

glycogenolysis and glucose output by the liver in the presence of basal intraportal

glucagon, high physiological insulin, and either euglycemia or hypoglycemia (6).

Moreover, it causes a dose-dependent stimulation of net hepatic glucose output (NHGO),

when the liver is in what normally would be a glucose uptake mode (in the presence of

hyperinsulinemia, a negative arterial-portal venous glucose gradient, and hyperglycemia)

(25). In light of the prevalence of insulin-induced hypoglycemia, it is notable that

AICAR is effective despite high physiological levels of insulin. The hypothesis tested in

the present study was that AICAR renders NHGO insensitive to a pharmacological

increase in insulin. To this end, AICAR was infused intraportally at rates 10% of those

used in rodent models (3, 4, 29) (to minimize extrahepatic effects) during euglycemic

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clamps in which somatostatin (SRIF) was infused, glucagon was replaced at basal rates,

and insulin was replaced at high physiological or pharmacological rates.

RESEARCH DESIGN AND METHODS

Animals and surgical procedures. Experiments were performed on a total of 19

overnight fasted mongrel dogs (mean wt. 22.2 ± 0.5 kg) of either sex that had been fed a

standard diet (7). Protocols were approved by the Vanderbilt University Animal Care

Committee. Surgical procedures used in this study have been previously described (7). Dogs

meeting the established health criteria (7) were studied after an 18 h overnight fast.

Experimental Procedures. Experiments consisted of tracer equilibration (-130 to -30

min), basal (-30 to 0 min), and hyperinsulinemic-euglycemic clamp (0 to 150 min) periods.

A primed (40 µCi), infusion (0.2 µCi•min-1) of high performance liquid chromatography

(HPLC) purified [3-3H]glucose was initiated at t =-130 min. The tracer infusion rate was

tripled at t=0 min. A SRIF infusion (0.8 µg•kg-1•min-1) was started at t=0 min to inhibit

insulin and glucagon secretion. Concurrent with SRIF infusion was basal intraportal

glucagon replacement (0.5 ng•kg-1•min-1). An intraportal insulin infusion of 2 (INS2, n=7) or

5 (INS5, n=12) mU•kg-1•min-1 was also initiated at this time. 6 dogs in INS2 comprise a

group published previously (6). A group of dogs treated identically to INS2 except without

AICAR from a previous publication (6) is included for comparison in Figures 1 and 3.

Arterial glucose was clamped at euglycemic levels (~105 mg•dL-1). A portal venous AICAR

infusion (1 mg•kg-1•min-1) was initiated at t=60 min. Portal vein and hepatic artery blood

flows were monitored on-line throughout the experiments. At the end of the experiment

animals were euthanized with sodium pentobarbital and liver biopsies were frozen in liquid

nitrogen. An autopsy was performed to confirm catheter placement.

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Blood sample and tissue collection and processing. Arterial, portal vein and hepatic

vein blood was drawn at t=-30, -15, 0, 15, 30, 45, 60, 75, 90, 120, and 150 min. Arterial

samples were taken every 10 min for the determination of plasma glucose specific activity.

Plasma glucose, glucose radioactivity, free fatty acids, cortisol, catecholamines, and

immunoreactive insulin and glucagon were determined as previously described (7). Blood

was deproteinized in perchloric acid for measurement of alanine, β-hydroxybutyrate,

glycerol, and lactate using standard enzymatic methods (18). Hepatic glycogen and purine

nucleotide concentrations were determined as previously described (6).

Western Blotting. 30 µg of protein from liver lysates (6) was run on a SDS-PAGE

gel and transferred to a polyvinyldine fluoride membrane. Membranes were treated with

rabbit α-phospho-AMP-activated protein kinase (AMPK, Thr172) or α-phospho-acetyl-CoA

carboxylase (ACC, Ser79) (Cell Signaling, Beverly, MA) and then incubated with α-rabbit-

HRP (Promega, Madison, WI). Chemiluminescence was detected with a WesternBreeze®

kit from Invitrogen (Carlsbad, CA). Densitometry was performed using Scion Image

software (Frederick, MD).

Calculations. Endogenous glucose production (Ra) and utilization (Rd) were

determined by isotope dilution (20).

Statistical Analysis. Statistical comparisons between groups and over time were

made using ANOVA designed to account for repeated measures. Specific time points were

examined for significance using contrasts solved by univariate repeated measures. Liver

purine nucleotides were compared using ANOVA designed to account for repeated measures.

Liver glycogen was compared using t-tests. AMPK and ACC phosphorylation was

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compared using one-tailed t-tests. Statistics are reported in tables and figures. Data are

presented as mean±SE. Statistical significance was defined as p<0.05.

RESULTS

Arterial plasma glucose and hormones. Arterial plasma glucose was similar during

the basal period and was clamped in both groups (107 ± 1 mg•dL-1 in INS2 and 104 ± 1

mg•dL-1 in INS5, Table 1). The glucose infusion required to clamp at euglycemia during the

last 90 min were 10.4 ± 0.5 mg•kg-1•min-1 and 16.4 ± 0.4 mg•kg-1•min-1 in INS2 and INS5,

respectively (Table 1). Arterial insulin rose to 52 ± 7 and 213 ± 26 µU•mL-1 in INS2 and

INS5, respectively (Table 1). Arterial plasma glucagon, cortisol, epinephrine, and

norepinephrine were similar between groups and did not change during the study (data not

shown).

Glucose kinetics. NHGO was similar during the basal period and fell similarly

during the first 60 min of hyperinsulinemia in each group (Figure 1). AICAR increased

NHGO similarly in INS2 (-0.2 ± 0.2 at t=60 min to 1.8 ± 0.6 mg•kg-1•min-1 at t=150 min,

p<0.05) and INS5 (-0.9 ± 0.3 at t=60 min to 1.1 ± 0.6 mg•kg-1•min-1 at t=150 min, p<0.05).

In contrast, NHGO remained suppressed when saline was infused instead of AICAR (Figure

1).

Endogenous Ra was similar during the basal period and fell during the first 60 min of

hyperinsulinemia in each group (Figure 2A). AICAR increased Ra in both INS2 (0.9 ± 0.2 at

t=60 min to 2.1 ± 0.7 mg•kg-1•min-1 at t=140 min) and INS5 (-1.7 ± 0.4 at t=60 min to 1.4 ±

0.6 mg•kg-1•min-1 at t=140 min, p<0.05). Rd was similar during the basal period and rose

similarly during the first 30 min of hyperinsulinemia in each group (Figure 2B). Rd was

significantly greater in INS5 compared to INS2 from t=40-140 min.

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Metabolite concentrations and net hepatic balances. Arterial concentrations and net

hepatic balances of alanine, β-hydroxybutyrate, free fatty acids, glycerol, and lactate were

similar between groups during the basal and hyperinsulinemia periods (data not shown).

Tissue analyses. At t=150 min, liver glycogen was 47 ± 4, 41 ± 3, and 35 ± 4 mg•g

liver-1 in a portal venous saline-infused group (from ref. (6)), INS2, and INS5, respectively

(p<0.05, INS5 vs. saline). Liver ZMP was not detectable, 3.51 ± 0.38, and 4.34 ± 0.46

µmol•g liver-1 compared to saline-infused dogs (from ref. (6)), INS2, and INS5, respectively

(p<0.05, INS2 and INS5 vs. saline). Phosphorylation of hepatic AMPK (Thr172) was similar

in all groups (data not shown). Phosphorylation of hepatic ACC (Ser79) was significantly

greater in INS2 versus INS5 and saline-infused dogs (p<0.05, Figure 3).

DISCUSSION

These studies show that intraportal infusion of AICAR at a rate of 1 mg•kg-1•min-1

renders hepatic glucose output blind to the suppressive effects of pharmacological insulin.

While it has previously been shown that intraportal AICAR stimulates hepatic glucose

output (6, 25), it was unknown if the effect was due to AMPK activation (and the

subsequent activation of glycogen-phosphorylase kinase (8) and –phosphorylase (31))

and/or allosteric activation of glycogen phosphorylase by ZMP (12, 19, 28). These studies

show that 1) AICAR is equally effective in stimulating hepatic glucose output at

physiological and pharmacological insulin levels and that 2) AICAR does not require an

increase in AMPK activity (as reflected by ACC phosphoylation) to exert this effect.

Several studies have examined the hepatic effects of administering AICAR in vivo.

Intracerebroventricular (ICV) infusion of AICAR increases endogenous Ra in 5 h fasted

mice (26) and overnight fasted rats (21). Iglesias et al. (16) showed that a 250 mg•kg-1

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intraperitoneal injection of AICAR caused a marked increment in net hepatic glycogen

breakdown in the 5 h fasted rat. Our laboratory has shown that in the presence of

hyperinsulinemia, basal glucagon, and either hypoglycemia, euglycemia (6), or

hyperglycemia (25), an intraportal AICAR infusion (1 mg•kg-1•min-1) increases hepatic

glucose output in the 18 h fasted dog. This AICAR infusion results in hepatic ZMP

concentrations of ~3 µmol·g liver-1, and an increase in hepatic AMPK activity, reflected by

an increase in hepatic ACC phosphorylation (6, 25). These studies were in contrast to those

of Bergeron et al., which showed that primed (100 mg·kg-1) systemic AICAR infusions of

7.5 (4) and 10 (3) mg•kg-1•min-1 suppressed Ra in overnight fasted rats. The differences

between the studies of Bergeron et al. and the previously mentioned studies showing an

AICAR stimulation of glucose production can be attributed to differences in glycogen

content, as an overnight fast virtually glycogen depletes rats. Consistent with differences in

hepatic glycogen levels being responsible for these differences is the fact that the marked

stimulation of NHGO from an intraportal AICAR infusion was lost when given to a 42 h

fasted dog in which liver glycogen was markedly reduced (R.C.C., D.H.W., unpublished

observation).

Intraportal AICAR infusion was associated with significant decreases in hepatic

glycogen (9 ± 4 and 12 ± 4 mg•g tissue-1 in INS2 and INS5, respectively). This coupled

with the fact that there were no differences in net hepatic alanine, glycerol, or lactate uptake

indicates that an increase in glycogenolysis was responsible for the rise in hepatic glucose

output. The differences in hepatic glycogen correspond to rates of net hepatic

glycogenolysis of 3.3 ± 1.2 and 4.7 ± 1.5 mg•kg-1•min-1 in INS2 and INS5, respectively.

Because endogenous Ra tends to be underestimated (as reflected by negative rates) under

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hyperinsulinemic-euglycemic clamp conditions (14), we focused on NHGO. However, it is

noteworthy that intraportal AICAR stimulated endogenous Ra to rise to a similar rate (~2

mg•kg-1•min-1) in both INS2 and INS5. Net hepatic glycogenolysis exceeded NHGO by

twofold, indicating that ~50% of the glucose-6-phosphate formed by glycogenolysis has a

fate other than glucose release. Since insulin stimulates pyruvate dehydrogenase, pyruvate

oxidation is likely to be an alternative fate (24).

The liver of the AICAR-infused dog appears to be resistant to the suppressive effects

of high physiological concentrations of insulin on hepatic glucose output (6, 25). This

suggests that a compound with similar liver actions could be effectively exploited to combat

insulin-induced hypoglycemia, which is the single most frequent complication in diabetic

patients (10). Both increased AMP/ZMP concentrations (12, 19, 28) and increased AMPK

activity (8, 31) have been shown to result in activation of glycogen phosphorylase. Even

small increments (11) in insulin potently inhibit glycogen phosphorylase in vivo (5) via its

regulation of cyclic AMP (13). This is one of the few studies to investigate insulin’s

regulation of hepatic AMPK.

ICV infusion of insulin reduces α2-AMPK activity in all hypothalamic regions in

mice (22). Chronic insulin treatment normalizes hypothalamic α2 AMPK hyperactivity in

STZ-induced diabetic rats (23). Beauloye et al. (2) showed that insulin antagonizes AMPK

activity (Thr172 phosphorylation) induced by ischemia or anoxia in rat hearts. Gamble and

Lospaschuk (15), as well as Witters and Kemp (30), have shown that AMPK inhibition and

ACC activation are equally sensitive to insulin, and that AMPK may be the major target for

insulin regulation of ACC. One way in which AMPK is activated, is by phosphorylation of

the Thr172 residue by an upstream kinase, AMPKK, or LKB1 (17). However, another study

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showed that AICAR does not activate AMPK by stimulating LKB1 (27). Given that AMPK

activity is regulated by both allosteric interactions as well as phosphorylation (9),

phosphorylation of ACC is a more sensitive marker of AMPK activity in vivo (1, 6, 25).

While there was no apparent increase in phosphorylation of the Thr172 residue of AMPK, the

downstream target of AMPK, ACC (Ser79) showed increased phosphorylation in the

presence of intraportal (physiological) insulin and AICAR infusions (Figure 3). This notion

is supported by other work that showed that AICAR treatment resulted in a clear increase in

the phosphorylation of ACC, but not in that of AMPK (19). The low AICAR dose used in

this study may not be high enough to cause covalent modification (phosphorylation) of

AMPK, yet may be sufficient to cause allosteric activation (19).

When the intraportal insulin infusion was increased to 5 mU•kg-1•min-1, ACC (Ser79)

phosphorylation was not different than that of saline-infused dogs, confirming that insulin

inhibits AMPK activation (2, 15, 22, 30). Since AMPK was not activated in the presence of

a 5 mU•kg-1•min-1 intraportal insulin infusion and the increase in glucose output remained, it

is clear that AICAR is stimulating net hepatic glycogen breakdown via an AMPK-

independent mechanism. This is consistent with the assertion that AICAR increases hepatic

glucose output in vivo (6, 25) via allosteric activation of glycogen phosphorylase (12, 19,

28).

In summary, results show that in the presence of basal glucagon, an intraportal

infusion of AICAR stimulates endogenous glucose output equally well regardless of

whether insulin concentrations are in the high physiological or pharmacological range. The

present results combined with earlier studies (6, 25) have numerous important implications.

First, because purine nucleotides are a natural constituent of all cells, including hepatocytes,

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they may comprise part of an endogenous pathway involved in physiological activation of

hepatic glucose output. Second, understanding hepatic insulin resistance caused by AICAR

may provide insight into basic mechanisms underlying this liver pathology. Third, AMPK-

independent actions of AICAR are a potent component of its glucoregulatory actions.

Fourth, mechanisms that increase hepatic purine nucleotide concentrations may be effective

in countering insulin-induced hypoglycemia. The fact that AICAR counters even

pharmacological levels of insulin, and that hypoglycemia potentiates the glycogenolytic

effect of AICAR (6) adds further weight to this last point. This suggests that a compound

with similar hepatic actions could effectively be exploited to combat insulin-induced

hypoglycemia, which is the single most frequently occurring complication in diabetes.

ACKNOWLEDGEMENTS

This work was funded by National Institute of Diabetes and Digestive and Kidney

Diseases Grant RO1 DK-50277, Diabetes Research and Training Center Grant DK-20593.

We would like to thank the Hormone Assay and Animal Resources Cores of the Vanderbilt

Diabetes Research and Training Center for providing the hormone and catecholamine data

provided.

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REFERENCES 1. Al-Khalili L, Krook A, Zierath JR, and Cartee GD. Prior Serum and AICAR-Induced AMPK Activation in Primary Human Myocytes Does Not Lead to Subsequent Increase in Insulin-Stimulated Glucose Uptake. Am J Physiol Endocrinol Metab, 2004.2. Beauloye C, Marsin AS, Bertrand L, Krause U, Hardie DG, Vanoverschelde JL, and Hue L. Insulin antagonizes AMP-activated protein kinase activation by ischemia or anoxia in rat hearts, without affecting total adenine nucleotides. FEBS Lett 505: 348-352, 2001. 3. Bergeron R, Previs SF, Cline GW, Perret P, Russell RR, 3rd, Young LH, and Shulman GI. Effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats. Diabetes 50: 1076-1082, 2001. 4. Bergeron R, Russell RR, 3rd, Young LH, Ren JM, Marcucci M, Lee A, and Shulman GI. Effect of AMPK activation on muscle glucose metabolism in conscious rats. Am J Physiol 276: E938-944, 1999. 5. Bishop JS, Goldberg ND, and Larner J. Insulin regulation of hepatic glycogen metabolism in the dog. Am J Physiol 220: 499-506, 1971. 6. Camacho RC, Pencek RR, James F, Lacy DB, and Wasserman DH. Portal venous 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside overcomes hyperinsulinemic suppression of endogenous glucose output. Diabetes 54: 373-382, 2005. 7. Camacho RC, Pencek RR, Lacy DB, James FD, and Wasserman DH. Suppression of endogenous glucose production by mild hyperinsulinemia during exercise is determined predominantly by portal venous insulin. Diabetes 53: 285-293, 2004. 8. Carling D and Hardie DG. The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase. Biochim Biophys Acta 1012: 81-86, 1989. 9. Corton JM, Gillespie JG, Hawley SA, and Hardie DG. 5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells? Eur J Biochem 229: 558-565, 1995. 10. Cryer PE. Hypoglycaemia: the limiting factor in the glycaemic management of Type I and Type II diabetes. Diabetologia 45: 937-948, 2002. 11. Edgerton DS, Cardin S, Emshwiller M, Neal D, Chandramouli V, Schumann WC, Landau BR, Rossetti L, and Cherrington AD. Small increases in insulin inhibit hepatic glucose production solely caused by an effect on glycogen metabolism. Diabetes 50:1872-1882, 2001. 12. Ercan-Fang N, Gannon MC, Rath VL, Treadway JL, Taylor MR, and Nuttall FQ. Integrated effects of multiple modulators on human liver glycogen phosphorylase a. Am J Physiol Endocrinol Metab 283: E29-37, 2002. 13. Exton JH, Lewis SB, Ho RJ, Robison GA, and Park CR. The role of cyclic AMP in the interaction of glucagon and insulin in the control of liver metabolism. Ann N Y Acad Sci 185: 85-100, 1971. 14. Finegood DT, Bergman RN, and Vranic M. Estimation of endogenous glucose production during hyperinsulinemic-euglycemic glucose clamps. Comparison of unlabeled and labeled exogenous glucose infusates. Diabetes 36: 914-924, 1987.

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15. Gamble J and Lopaschuk GD. Insulin inhibition of 5' adenosine monophosphate-activated protein kinase in the heart results in activation of acetyl coenzyme A carboxylase and inhibition of fatty acid oxidation. Metabolism 46: 1270-1274, 1997. 16. Iglesias MA, Ye JM, Frangioudakis G, Saha AK, Tomas E, Ruderman NB, Cooney GJ, and Kraegen EW. AICAR administration causes an apparent enhancement of muscle and liver insulin action in insulin-resistant high-fat-fed rats. Diabetes 51: 2886-2894, 2002. 17. Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J, Hawley SA, Udd L, Makela TP, Hardie DG, and Alessi DR. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. Embo J 23: 833-843, 2004. 18. Lloyd B, Burrin J, Smythe P, and Alberti KG. Enzymatic fluorometric continuous-flow assays for blood glucose, lactate, pyruvate, alanine, glycerol, and 3-hydroxybutyrate. Clin Chem 24: 1724-1729, 1978. 19. Longnus SL, Wambolt RB, Parsons HL, Brownsey RW, and Allard MF. 5-Aminoimidazole-4-carboxamide 1-beta -D-ribofuranoside (AICAR) stimulates myocardial glycogenolysis by allosteric mechanisms. Am J Physiol Regul Integr Comp Physiol 284:R936-944, 2003. 20. Mari A. Estimation of the rate of appearance in the non-steady state with a two-compartment model. Am J Physiol 263: E400-E415, 1992. 21. McCrimmon RJ, Fan X, Ding Y, Zhu W, Jacob RJ, and Sherwin RS. Potential role for AMP-activated protein kinase in hypoglycemia sensing in the ventromedial hypothalamus. Diabetes 53: 1953-1958, 2004. 22. Minokoshi Y, Alquier T, Furukawa N, Kim YB, Lee A, Xue B, Mu J, Foufelle F, Ferre P, Birnbaum MJ, Stuck BJ, and Kahn BB. AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus. Nature 428: 569-574, 2004. 23. Namkoong C, Kim MS, Jang PG, Han SM, Park HS, Koh EH, Lee WJ, Kim JY, Park IS, Park JY, and Lee KU. Enhanced hypothalamic AMP-activated protein kinase activity contributes to hyperphagia in diabetic rats. Diabetes 54: 63-68, 2005. 24. Parker JC and Jarett L. Insulin mediator stimulates pyruvate dehydrogenase of intact liver mitochondria. Diabetes 34: 92-97, 1985. 25. Pencek RR, Shearer J, Camacho RC, James FD, Lacy DB, Fueger PT, and Wasserman DH. 5'-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside causes acute hepatic insulin resistance in vivo. Diabetes 54: 355-360, 2005. 26. Perrin C, Knauf C, and Burcelin R. Intracerebroventricular infusion of glucose, insulin and the AMP-activated kinase activator AICAR controls muscle glycogen synthesis. Endocrinology 145: 4025-4033, 2004. 27. Sakamoto K, Goransson O, Hardie DG, and Alessi DR. Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR. Am J Physiol Endocrinol Metab 287: E310-317, 2004. 28. Shang J and Lehrman MA. Activation of glycogen phosphorylase with 5-aminoimidazole-4-carboxamide riboside (AICAR). Assessment of glycogen as a precursor of mannosyl residues in glycoconjugates. J Biol Chem 279: 12076-12080, 2004.

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29. Shearer J, Fueger PT, Vorndick B, Bracy DP, Rottman JN, Clanton JA, and Wasserman DH. AMP Kinase-Induced Skeletal Muscle Glucose But Not Long-Chain Fatty Acid Uptake Is Dependent on Nitric Oxide. Diabetes 53: 1429-1435, 2004. 30. Witters LA and Kemp BE. Insulin activation of acetyl-CoA carboxylase accompanied by inhibition of the 5'-AMP-activated protein kinase. J Biol Chem 267: 2864-2867, 1992. 31. Young ME, Radda GK, and Leighton B. Activation of glycogen phosphorylase and glycogenolysis in rat skeletal muscle by AICAR - an activator of AMP-activated protein kinase. FEBS Lett 382: 43-47, 1996.

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Figure Legends Figure 1: Net hepatic glucose output in the presence of a 2 mU•kg-1•min-1 (n=7, closed

circles) and 5 mU•kg-1•min-1 (n=12, open circles) hyperinsulinemic infusion during the basal

period (-30 to 0 min) and hyperinsulinemic-euglycemic clamp period (0 to 150 min) in the

presence of intraportal AICAR from 60 to 150 min. The dotted line represents the normal

response of net hepatic glucose output in the presence of hyperinsulinemia alone (data from

ref. (6)). Inset graph shows area under the curve (AUC) of NHGO after t=60 min. Data are

mean ± SE. *Significantly different than SALINE (p<0.05).

Figure 2: A) Endogenous Ra and B) Rd in the presence of a 2 mU•kg-1•min-1 (n=7, closed

circles) and 5 mU•kg-1•min-1 (n=12, open circles) hyperinsulinemic infusion during the basal

period (-30 to 0 min) and hyperinsulinemic-euglycemic clamp period (0 to 150 min) in the

presence of intraportal AICAR from 60 to 150 min. Data are mean ± SE. *Significantly

different than 5 mU•kg-1•min-1 (p<0.05).

Figure 3: Quantification of the phosphorylated form of ACC (Ser79) from livers after a 2

mU•kg-1•min-1 hyperinsulinemic infusion with a portal venous SALINE infusion (n=5, grey

bar, from ref. (6)), and portal venous AICAR treated dogs in the presence of a 2 mU•kg-

1•min-1 (n=7, black bar) and 5 mU•kg-1•min-1 (n=5, open bar). Data are mean ± SE.

*Significantly different than SALINE and INS5 (p<0.05).

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Time (min)-30 0 30 60 90 120 150

NH

GO

(mg·

kg-1

·min

-1)

-2

-1

0

1

2

3

4

2 mU·kg-1·min-1 5 mU·kg-1·min-1

Hyperinsulinemia

AICAR

2 mU·kg-1·min-1

Insulin, noAICAR

SALINE INS2 INS5

NH

GO

AUC

(mg·

kg-1)

0

20

40

60

80

100

120 **

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Endo

geno

usR

a(m

g·kg

-1·m

in-1

)

-3

-2

-1

0

1

2

3

4

Time (min)-30 0 30 60 90 120 150

Rd

(mg·

kg-1

·min

-1)

0

5

10

15

20

25

2 mU·kg-1·min-1

5 mU·kg-1·min-1

* * * ** * * * *

Hyperinsulinemia

AICAR

**

A

B

* ** *

*

* *

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ACC

Ser79

Phos

phor

ylat

ion

(Arb

itrar

yU

nits

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

INS2 INS5SAL

*

$ASQE-00247-2005.R1

20

Table 1. Arterial plasma glucose, insulin, and glucose infusion rates during baseline and

hyperinsulinemic-euglycemic clamp periods in the presence and absence of intraportal AICAR.

Data are mean ± SE (INS2, n=7; INS5, n=12). *p<0.05 vs. INS2.

Hyperinsulinemia

Intraportal AICAR

Basal 15 30 60 90 120 150

Arterial Plasma Glucose (mg•dL-1)INS2 111 ± 1 107 ± 3 107 ± 3 106 ± 2 108 ± 2 106 ± 3 110 ± 3 INS5 110 ± 1 104 ± 4 103 ± 3 102 ± 2 110 ± 2 103 ± 2 105 ± 1

Arterial Plasma Insulin (µU•mL-1)INS2 6 ± 0 37± 5 41 ± 8 42 ± 8 45 ± 8 49 ± 7 52 ± 7 INS5 8 ± 1 141± 17* 157 ± 20* 171 ± 24* 187 ± 28* 197 ± 23* 213 ± 26*

Glucose Infusion Rate (mg•kg-1•min-1)INS2 – 3.3 ± 0.8 5.7 ± 1.0 7.9 ± 1.2 9.8 ± 1.4 11.3 ± 1.5 11.7 ± 1.3 INS5 – 5.1 ± 0.5* 8.9 ± 1.8* 15.8 ± 1.2* 18.4 ± 1.8* 16.9 ± 1.2* 17.3 ± 1.2*